Biology subjects

Parekkadan, B.

Publications and source records attributed to Parekkadan, B..

7 recordsLinked to original sources

bioRxiv2025

Cutaneous suction-mediated transfection in mice for delivery of DNA-encoded vaccines and proteins

An important step to fulfill the functionalities of DNA vaccines and therapeutics is transfection in vivo to produce the encoded antigens or therapeutic proteins. A cutaneous suction-based method has demonstrated effectiveness in many animal models and has been successfully applied in human clinical trials, but has not been extended to mouse models, where numerous disease models, transgenic strains, and murine-specific reagents exist. The current work establishes and optimizes methods for cutaneous suction-mediated DNA transfection in mice. By adapting a smaller cup diameter and smaller injection volume, the challenges of skin hyperelasticity and decreased skin thickness can be effectively addressed, and vaccinating mice with the GLS-5310 SARS-CoV-2 DNA vaccine yielded high levels of binding antibody and T cell responses. Additionally, suction following injection of a novel pVAX1-based expression vector yielded systemic levels of a SEAP transgene. Thus, suction-mediated delivery of nucleic acid-based therapies and vaccines can be a valuable tool for the study in pre-clinical mouse models.

bioengineering↗

bioRxiv2023

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

synthetic biology↗

bioRxiv2023

Simulating Interclonal Interactions in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of cancers, accounting for 37% of B-cell tumors globally. DLBCL is known to be a heterogeneous disease, resulting in variable clinical presentations and the development of drug resistance. One underexplored aspect of drug resistance is the evolving dynamics between parental and drug-resistant clones with the same microenvironment. In this work, the effects of interclonal interactions between two cell populations - one sensitive to treatment and another resistant to treatment - on tumor growth behaviors were explored through a mathematical model. In vitro cultures of mixed DLBCL populations demonstrated cooperative interactions and revealed the need for modifying the model to account for complex interactions. Multiple best-fit models derived from in vitro data indicated a difference in steady-state behaviors based on therapy administrations in simulations. The model and methods may serve as a tool in understanding the behaviors of heterogeneous tumors and in identifying the optimal therapeutic regimen to eliminate cancer cell populations using computer-guided simulations. ImportanceThe cellular makeup of tumors can play a vital role in its growth and cancer development. In this work, two different types of cell populations of diffuse large B-cell lymphoma (DLBCL) were studied together to understand how they interact with each other in cultures. In mixed cultures, both types of cells cooperated with each other and increased their growth in complex manners. A mathematical model was created to simulate the growth behavior of mixed cultures. The model can potentially be used to predict future cell behavior and help in identifying more effective therapy regimens to maximize tumor cell reduction.

cancer biology↗

bioRxiv2023

Capture Efficiency Of Long-Adapter Single-Strand Oligonucleotide Probe Libraries

High throughput techniques that can massively produce in parallel, longer DNA sequences of interest can accelerate the decoding of gene functions. LASSO probes are a molecular biology tool that can enrich for DNA targets in a genomic sample via a multiplexed, single-pot reaction for downstream sequencing and/or cloning. Here we have explored aspects of process development and the design of the probes that relate to binding thermodynamics to determine impact on cloned library sequences. Control of ligase concentration, polymerase type, and melting temperature of probe are critical when translating the use of LASSO probes for homogeneous and high fidelity DNA capture.

genomics↗

bioRxiv2023

3D Microcapsules for Human Bone Marrow Derived Mesenchymal Stem Cell Biomanufacturing in a Vertical-Wheel Bioreactor

Microencapsulation of human mesenchymal stromal cells (MSCs) via electrospraying has been well documented in tissue engineering and regenerative medicine. Herein, we report the use of microencapsulation, via electrospraying, for MSC expansion using a commercially available hydrogel that is durable, optimized to MSC culture, and enzymatically degradable for cell recovery. Critical parameters of the electrospraying encapsulation process such as seeding density, correlation of microcapsule output with hydrogel volume, and applied voltage were characterized to consistently fabricate cell-laden microcapsules of uniform size. Upon encapsulation, we then verified [~] 10x expansion of encapsulated MSCs within a vertical-wheel bioreactor and the preservation of critical quality attributes such as immunophenotype and multipotency after expansion and cell recovery. Finally, we highlight the genetic manipulation of encapsulated MSCs as an example of incorporating bioactive agents in the capsule material to create new compositions of MSCs with altered phenotypes.

bioengineering↗

bioRxiv2021

Neutralizing IFNL3 Autoantibodies in Severe COVID-19 Identified Using Molecular Indexing of Proteins by Self-Assembly

Unbiased antibody profiling can identify the targets of an immune reaction. A number of likely pathogenic autoreactive antibodies have been associated with life-threatening SARS-CoV-2 infection; yet, many additional autoantibodies likely remain unknown. Here we present Molecular Indexing of Proteins by Self Assembly (MIPSA), a technique that produces ORFeome-scale libraries of proteins covalently coupled to uniquely identifying DNA barcodes for analysis by sequencing. We used MIPSA to profile circulating autoantibodies from 55 patients with severe COVID-19 against 11,076 DNA-barcoded proteins of the human ORFeome library. MIPSA identified previously known autoreactivities, and also detected undescribed neutralizing interferon lambda 3 (IFN-{lambda}3) autoantibodies. At-risk individuals with anti-IFN-{lambda}3 antibodies may benefit from interferon supplementation therapies, such as those currently undergoing clinical evaluation. One-Sentence SummaryMolecular Indexing of Proteins by Self Assembly (MIPSA) identifies neutralizing IFNL3 autoantibodies in patients with severe COVID-19. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/432977v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@a3c55aorg.highwire.dtl.DTLVardef@1f1c840org.highwire.dtl.DTLVardef@920bc7org.highwire.dtl.DTLVardef@43633e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Preclinical Mesenchymal Stromal Cell Delivery Via An Ex Vivo Bioreactor Attenuates Clot Formation For Intravascular Applications

While mesenchymal stromal cells (MSCs) are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel ex vivo approach aimed at improving the safety of MSC systemic administration by use of a bioreactor. In this device, MSCs are seeded on the outside of a hollow-fiber filter, sequestering them behind a hemocompatible membrane, while still maintaining cross talk with blood cells and circulating signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against "free" MSCs, as a model of systemic administration, which were directly injected into the circuit. Our results showed that physical isolation of the MSCs via a bioreactor extends the time necessary for clot formation to occur when compared to "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Further, application of this technology in two in vivo models did not require additional heparin to maintain target ACT levels relative to the acellular device. Taken together, the use of hollow fiber filters to house MSCs, if adopted clinically, could offer a novel method to control systemic MSC exposure and prolong clot formation time.

bioengineering↗